A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

How does caspases regulation play role in cell decisions? apoptosis and beyond.

Caspases are a family of cysteine proteases, and the key factors behind the cellular events which occur during apoptosis and inflammation. However, increasing evidence shows the non-conventional pro-survival action of apoptotic caspases in crucial processes. These cellular events include cell proliferation, differentiation, and migration, which may appear in the form of metastasis, and chemotherapy resistance in cancerous situations. Therefore, there should be a precise and strict control of caspases activity, perhaps through maintaining the threshold below the required levels for apoptosis. Thus, understanding the regulators of caspase activities that render apoptotic caspases as non-apoptotic is of paramount importance both mechanistically and clinically. Furthermore, the functions of apoptotic caspases are affected by numerous post-translational modifications. In the present mini-review, we highlight the various mechanisms that directly impact caspases with respect to their anti- or non-apoptotic functions. In this regard, post-translational modifications (PTMs), isoforms, subcellular localization, transient activity, substrate availability, substrate selection, and interaction-mediated regulations are discussed.